The present invention concerns the area of assaying chemical or biological substances, such as sequences of nucleic acids or proteins by specific reaction with a set of specific reagents fixed on a support. It relates more particularly to the assay of nucleic acids by hybridisation with oligonucleotide probes fixed on a support. This type of assay finds application in sequencing, diagnosis or quality control for research or industrial production.
The match law A-T (or A-U) and G-C gives nucleic acids the property of forming specific hybridisation complexes between complementary sequences. This long known hybridisation property means that a fragment of nucleic acid, or an oligonucleotide, can be used to display the presence of a complementary nucleic sequence. The analysis of DNA fragments after gel separation (E. Southern, J. mol. Biol., 1975, 98, 503-517) is today largely used both in the area of fundamental research and in medical analysis (Caskey, Science, 1987, 236, 1223-1228; Landegrer et al., Science, 1988, 242, 229-237; Arnheim et al., Ann rev. Biochem., 1992, 61, 131-156).
Several assay techniques of nucleic acids based on their hybridisation property with probes have been developed, in particular for the sequencing of unknown DNAs or RNAs, the detection of sequences associated with a pathology, and the search for point mutations in sequences (S. Ikata et al., Nuclei Acids Res., 1987, 15, 797-811; J. A. Matthews, L. J. Krieka, Analytical Biochemistry, 1988, 169, 1-25).
More recently, new assay methods for unknown DNA fragments have been put forward, based on hybridisation with a series of oligonucleotides comprising 5 to 9 bases arranged in known sequence and immobilised on a solid support (E. Southern, European Patent published under number: 0 373 203; K. R. Khrapko et al., FEBS Letters, 1989, 256, 118-122 ; R. Drmanac et al., Genomics, 1989, 4, 114-128 ; R. Drmanac et al., DNA and Cell Biology, 1990, 9, 527-534 ; K. R. Khrapko et al., J. DNA Sequencing Mapp., 1991, 1, 375-388 ; R. Drmanac et al., Science 1993, 260, 1649-1652 ; R. J. Lipshutz, J. Biomol. Struct. Dyn., 1993, 11, 637-653; U. Maskos, E. Southern, Nucleic Acids Res., 1993, 21, 4663-4669 ; A. C. Pease et al., Proc. Natl. Acad. Sci., USA, 1994, 91, 5022-5026; J. C. Williams, Nucleic Acids Res., 1994, 22, 1365-1367 ; E. M. Southern, Nucleic Acids Res., 1994, 22, 1368-1373.
These new assay methods are based on the preparation of a support on whose surface is fixed a series of oligonucleotide sequences having the same length covering all possible sequences for this length, each of the oligonucleotide sequences occupying cells, or zones, separated on the support. The preparation and use of said support is described in particular in European patent applications published under Nxc2x0s 373 203 and 392 546 and in international patent application published under nxc2x0 WO 94/12670. The sequence of nucleic acid to be assayed is labelled and applied to the support under conditions allowing hybridisation with the oligonucleotides, then after washing, the localisation of the labelling is noted on the support surface in order to detect the signals transmitted by any hybrids which may be formed between the assayed, labelled DNA fragment and one or more oligonucleotides of the series immobilised on the solid support. Each position shown to be positive on the support corresponds to a complementary sequence thus identified in the assayed DNA fragment.
Immobilisation of the oligonucloetides on the support surface may be conducted in accordance with two principles: either immobilisation after synthesis of the oligonucleotides, or direct synthesis of the oligonucleotides on the support.
Immobilisation after synthesis of the oligonucleotides is difficult to implement and does not appear sufficiently reproducible as is the case for antibodies ( European patent Nxc2x0 0 063 810), therefore most teams working on this subject have chosen to develop techniques for direct synthesis on the support to be used for hybridisation and detection (International Patent Applications WO 90 03 382, WO 90 15 070, WO 91 08 307, WO 91 07 087, WO 92 10 092, WO 92 10 587, WO 93 10 161, WO 93 09 668, WO 94 22 889, WO 95 11 748, WO 95 30 774). These involve the synthesis one same surface of a series of oligonucleotide sequences having the same length covering all possible sequences for this length. Therefore, for oligonucleotides with 2 bases, chosen from among the four forming the DNA, 16 sequences are possible, for oligonucleotides with 3 bases 64 sequences are possible, for oligonucleotides with 4 bases 256 sequences are possible, for oligonucleotides with 5 bases 1024 sequences are possible, for oligonucleotides with 6 bases 4096 sequences are possible etc.. The shorter the oligonucleotide sequence, the less information it provides for sequencing; for a DNA fragment with arrangement, all or part of said homogeneous supports are mutually combined to form assay supports each having an ordered set of reaction zones of N different types or less.
By xe2x80x9ctype of reaction xe2x80x9d is meant the specific reaction likely to occur between the assayed substance and the support, when they are brought into contact. This reaction is determined by the nature of the different N reagents, present in ordered and verifiable manner on the supports obtained by the method of the invention, each of the initial N supports being homogeneous for one of its reagents. For the assay of nucleic acids, by type of reaction is meant the specific hybridisation reaction likely to occur between an oligonucleotide sequence fixed on one of the initial N supports, re-detected in a reaction zone on the final support, and a complementary nucleic acid sequence present in the assayed substance.
According to one first particular embodiment, the method of the invention comprises the following stages:
a) preparation of N flat shaped supports homogeneous for a type of reaction;
b) cutting said flat, homogeneous N supports into strips of substantially equal width;
c) combining substantially side by side the strips derived from at least one part of the N homogeneous supports so as to prepare intermediate supports which, along an axis that is perpendicular to said strips of homogeneous supports, comprise alignments of different reaction zones, each of said alignments of one same intermediate support being identical;
d) cutting the intermediate supports into strips of substantially the same width, each of said strips comprising one of said alignments of different reaction zones,
e) combining substantially side by side the strips which each comprise an alignment of different reaction zones, in such manner as to form assay supports each having alignments of ordered reaction zones of N different types or less.
According to a second particular embodiment, the method of the invention comprises the following stages:
axe2x80x2) preparation of N homogeneous supports in the form of filaments or fibres, and
bxe2x80x2) combining substantially side by side several of said filaments in such manner as to prepare intermediate supports which, along an axis perpendicular to said filaments, comprise alignments of different reaction zones, each of said alignments of one same intermediate support being identical, then, carrying out stages (d) and (e) previously described in the first embodiment.
Stage (bxe2x80x2) above may consist of gluing side by side the homogeneous filaments prepared in stage (axe2x80x2) so as to form the intermediate supports defined in stage (c) of the first embodiment.
One variant of stages (a) and (axe2x80x2) described above, consists of:
axe2x80x3) preparing N homogeneous supports in the form of filaments, and proceeding with weaving each of said N filaments with a weft made of reactively neutral strand so as to prepare the N homogeneous supports of the type of those in stage (a) described in the first embodiment,
then, carrying out stages (b), (c), (d) and (e) previously described.
One variant of stages (axe2x80x3) and (bxe2x80x2) described above consists of:
axe2x80x2) preparing N homogeneous supports in the form of filaments, and
bxe2x80x3) proceeding with weaving all or part of the homogeneous N filaments by intertwining a weft made of said N filaments with a strand that is reactively neutral, so as to prepare intermediate supports or a final support, then
optionally carrying out stages (d) and (e) described previously, if the supports prepared at stage (bxe2x80x3) are intermediate supports.
In order to maintain the rigidity of the intermediate support of stages (b), (bxe2x80x2)and (bxe2x80x3), it is advantageously fixed to an auxiliary element for temporary or permanent support; xe2x80x9ctemporaryxe2x80x9d meaning that the auxiliary support element is removed after stage (e) or (bxe2x80x3), xe2x80x9cpermanentxe2x80x9d meaning that the auxiliary support element is left in place after stage (e) or (bxe2x80x3).
Also, the final support obtained at stages (e) and (bxe2x80x3) may advantageously be made rigid and therefore handled with greater ease if it is fixed onto a support element. Also, this support element may be used to give a particular shape to the final support, such as for example one or more recesses to facilitate and border the deposits of hybridisation and revealing reagents at each reaction zone, or even to form an edge for the support.
The homogeneous supports of flat shape or in fibre form may be in a porous or non-porous matter. As porous flat support, preference is given to a cellulose microporous membrane.
If the support of the invention is intended to be used for the assay of nucleic acids, the initial N supports are each homogeneous for one type of specific hybridisation reaction. Advantageously, each of the N homogeneous supports then comprises a different oligonucleotide sequence. In one particular embodiment, intended for the sequencing or assay of mutations, on each of the N homogeneous supports is fixed a different oligonucleotide sequence but of similar length, all the oligonucleotide sequences of the N supports then covering all the possible sequences for this length.
Assay of the extent of hybridisation with each of the oligonucleotides, for example using the Tm values, at each reaction zone of the supports, can determine with great precision the sequence of the assayed nucleic acid, DNA or RNA or the presence of a mutation.
The supports of the inventions may also be used for immunological assay; in this case, the N supports are each homogeneous for one type of antigen-antibody reaction. In this embodiment, one different antibody or one antigen, according to whether the assayed substance is an antigen or an antibody, is fixed by any method known to men of the art on each of the initial N supports.
The supports of the invention may also be used for the assay of substrates likely to react with a series of different enzymes each placed at the level of each reaction zone.
The invention also relates to an assay support prepared using the above-described methods, and the use of this support for methods of assaying chemical or biological substances, and its incorporation into assay kits for chemical or biological substances.
Other characteristics and advantages of the invention will become apparent on reading the following examples which are given for guidance purposes and are not restrictive.